Dr. Laue is a clinical pediatric endocrinologist with experience as a principal investigator on numerous clinical research protocols. Her major clinical focus has been to pioneer treatment for boys with familial male precocious puberty (FMPP, a gonadotropin-independent disorder, characterized by Leydig cell hyperplasia, increased testosterone production, and prepubertal levels of luteinizing hormone [LH]). Recently, Dr. Laue has identified a substitution of glycine for aspartate578 and glycine for aspartate564 in the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor in males with FMPP. Expression of these mutant receptors in COS-7 and 293 cells resulted in increased levels of basal cAMP, consistent with constitutive activation of the receptor. The objectives of this project are to identify other (constitutively activating) mutations of the LH/CG receptor in individuals with FMPP, to define the mechanism(s) by which such mutations cause human disease/pathology, and to design new treatments for FMPP using "code blockers" to selectively interrupt gene expression. The specific aims of this project are: 1) To identify new mutations of the LH/CG receptor in patients with FMPP using restriction endonuclease analysis, PCR-based mutation analysis, and direct PCR sequencing of genomic DNA,. 2) To confirm that the Asp578->Gly and Asp564->Gly mutations result in Leydig cell hyperplasia and increased steroidogenesis in vitro through a cAMP-dependent mechanism. MA-10, MA-10(K3), and Y-1 cells expressing the wild type and/or mutant LH/CG receptors will be used to study the effects of endogenous cAMP, cAMP analogs, adenylate cyclase antagonists, and protein kinase A inhibitors on steroidogenesis and DNA synthesis. 3) To determine cAMP-dependent or -independent effects of the Asp578->Gly and Asp564->Gly mutations on phospholipase C activation in vitro. MA-10, and Y-1 cells expressing the wild type and/or mutant LH/CG receptors will be used to quantitate inositol phosphate generation in the presence or absence of adenylate cyclase antagonists and phospholipase C inhibitors. 4) To determine the mechanism whereby Asp578->Gly and Asp564->Gly effects LH/CG receptor activity, through site-directed mutagenesis, expression studies. Dr. Chan's laboratory in conjunction with the macromolecular core facility of the Lombardi Cancer Center will provide all necessary resources and equipment to perform the molecular studies outlined in Dr. Laue's research plan.